Apoptosis is now recognized as an essential biological process in tissue homeostasis of all living species. In mammals in particular, it has been shown to regulate embryonic development. Later in life, cell death is a default mechanism that removes potentially dangerous cells (e.g. cells carrying cancerous defects). Several apoptotic pathways have been uncovered and one of the most important involves the Bcl-2 family of proteins. The structural homology domains BH1 to BH4 are characteristic of this family. Further classification into of three subfamilies depends on how many of these homology domains a protein contains and on its biological activity (pro- or anti-apoptotic).
The first subgroup contains proteins having all 4 homology domains BH1 to BH4. Their general effect is anti-apoptotic thus preserving the cell from starting a cell death process. Proteins such as Bcl-2, Bcl-w and BCl-xL are members of this first subgroup. Proteins belonging to the second subgroup have a pro-apoptotic effect and contain the three homology domains BH1 to BH3. The two main representative proteins of this second subgroup are Bax and Bak. Finally, the third subgroup is composed of protein containing only the BH3 domain and members of this subgroup are usually referred to as “BH3-only proteins”. Their biological effect on the cell is pro-apoptotic. Bim, Bad, Bmf, and Bid are examples of this third subfamily of proteins.
The delicate balance between the three subgroups is the key to homeostasis of the cells. Recent studies have tried to elucidate the mechanisms involving the Bcl-2 family of proteins that allow a cell to undergo programmed cell death upon receiving intra- or extra-cellular signal. Such a signal induces the activation (post translational or transcriptional) of BH3-only proteins. These proteins are the primary inducers of the cascade that leads to cell death. The BH3-only proteins mainly interact with the Bcl-2 subgroup and stop proteins such as Bcl-2, BCl-xL or Bcl-w from inhibiting the Bax/Bak subgroup. These later proteins are either already anchored to the mitochondrial membrane or migrate to this membrane. Their activation leads to membrane swelling, release of cytochrome C and downstream activation of effector caspases resulting in apoptosis.
As already mentioned the balance between these proteins is essential to the correct cellular response to various stimuli. Any perturbation of this balance will instigate or worsen major diseases. Thus apoptosis perturbations have been shown to be at the origin of important diseases such as neurodegenerative conditions (up-regulated apoptosis) for example, Alzheimer's disease, or proliferative diseases (down-regulated apoptosis) for example, cancer and autoimmune diseases.
The discovery that several proteins of the Bcl-2 family are involved in the onset of cancerous malignancy has unveiled a completely novel way of targeting this still elusive disease. It has been shown in particular that pro-survival proteins such as Bcl-2 are over-expressed in many cancer types (see Table 1) [Zhang, 2002]. The effect of this deregulation is the survival of altered cells which would have undergone apoptosis in normal conditions. The repetition of these defects associated with unregulated proliferation is thought to be the starting point of cancerous evolution. BH3-only proteins have also been shown to act as tumor suppressors when expressed in diseased animals.
TABLE 1Bcl-2 over-expression in cancerCancer typeBcl-2 over-expressionHormone-refractory90-100%prostate cancerMalignant melanoma90%Oestrogen-receptor-80-90%positive breast cancerNon-Hodgkin's50%lymphomaColon Cancer30-50%Chronic lymphocytic25-50%leukaemia
These findings as well as numerous others have made possible the emergence of new concept in anti-cancer strategies and drug discovery. If an entity mimicking the effect of BH3-only proteins were able to enter the cell and overcome the pro-survival protein over-expression, it could be possible to reset the apoptotic process. This strategy may have the advantage that it may alleviate the problem of drug resistance which is usually a consequence of apoptotic deregulation (abnormal survival).
A considerable effort has been made to understand the structural details of the key interactions between BH3-only proteins and the pro-survival subgroup. Fesik and co-workers have demonstrated in the case of the dimer Bad/Bcl-xL the importance of some structural elements [Muchmore et. al., 1996; Sattler et. al., 1997 and Petros et. al., 2000]:                binding occurs between a hydrophobic groove located on BCl-xL and the BH3 domain of Bad;        the BH3-only protein Bad adopts a helix structure upon binding to the hydrophobic groove of BCl-xL; and        four hydrophobic amino-acids of the BH3 domain located at i, i+3, i+7 and i+11 intervals are essential to the binding of Bad to BCl-xL and interact in four hydrophobic pockets situated in the BCl-xL binding groove. Moreover, studies of members of the BH3-only subgroups have shown that these four hydrophobic amino-acids are conserved through the subgroup.        
Recently the structure of the pro-survival protein Bcl-w [Hinds et. al., 2003] and the structure of BH3-only protein Bim in interaction with BCl-xL [Liu et. al., 2003] have been published. This latter structure confirms the findings of the Bad/Bcl-xL interaction.
A potential target for new drug therapy is small molecules that mimic the interaction between a BH3-only protein and the Bcl-2 family of proteins. Recently a small molecule BH3-only protein mimetic has been shown to have cytotoxic activity in some cancer cell lines and to enhance the effects of radiation therapy and a number of chemotherapeutic agents [Oltersdorf et. al., 2005; US 2002/0086887; WO 03/080586; U.S. Pat. No. 6,720,338; WO 05/049597; Petros, et al., 2006; Cory and Adams, 2005].
The alpha-helix is a common recognition motif displayed in peptides and proteins. Alpha-helical sequences are often involved in protein-protein interactions, such as enzyme-receptor and antibody-receptor interactions. Targeting these protein-protein interactions is now recognised as one of the major challenges in drug discovery.
There is a need for small molecules which may be easily synthesised and that mimic the activity of BH3-only proteins.